PROJECTSUMMARY Interactionsbetweengutbacteriaandintestinalcellshavebeenimplicatedinmanyhuman diseasesincludinginflammatoryboweldisease,diabetes,metabolicsyndrome,andmanymore. Currently,gnotobioticanimalmodelsarethegoldstandardfortestingandunderstandingthese interactions,butanimalexperimentsarecostlyandgnotibioticcoloniesrequirelargeamountsof resources.Weproposethatculturedintestinalorganoidsorminigutscanbeusedasan intermediateenvironmentinwhichbacteriacanbeculturedandtheeffectofbacterialculture andsecretiononmammaliangeneexpressioncanbemonitoredatrelativelyshortertimesand lowercosts.Ourapproachistoco-cultureengineeredprobioticbacteriatoaccuratelymonitor andactivelyinfluencethedevelopmentofintestinalepithelialcellsin3Dculture. WeproposetodevelopthistechnologyviathefollowingSpecificAims: Aim1:Establishreal-timephenotypiccharacterizationofcelldifferentiationinminiguts byengineeredwhole-cellbiosensors.Wewilldevelopwholecellbiosensorsforintestinal secretionssuchaschlorideandserotoninusingengineeredprobioticE.colibacteria.The rationalebehindthisaimisthatsecretionofthesemoleculesisanindicationofproper differentiationofintestinalstemcellsintodistinctcelltypes.Thebiosensorwithfluorescent outputwilloperateinreal-timewhichwillallowdynamicvisualizationofsecretionphenotypesby fluorescencemicroscopy. Aim2:Alterminigutgrowthbysecretionofsmallmoleculesandgrowthfactorsfrom commensalbacteria.Wewillengineerprobioticbacteriatodelivermoleculesthatnegatively andpositivelyinfluencetheproliferationofstemcellsin3Dculture.Therationaleofthisaimis thatsecretionofbutyrate,akeymetabolite,isnaturallymicrobially-driveninthegutandthe effectofmicrobialbutyrateproductionongutcellsisprofound.Inaddition,wewishtostudy positiveregulationofgrowthbybacterialsecretionthegrowthfactorR-spondin-1intheminigut lumen. Takentogether,successfulimplementationoftheseaimswilldeveloparobustco-culture platformfortheoptimizationof3Dorganoidculturesandthestudyofhost-microbeinteractions.